Leishmania spp. is innately endowed with exceptionally favorable attributes for exploitation as a potential universal vaccine carrier. Transgenic biotechnology is well-developed for these eukaryotic protozoa to express foreign proteins for vaccination. In addition, adjuvanticity of Leishmania on vaccination is evident from the observation that life-long lasting immunity ensue invariably after spontaneous cure of human simple cutaneous leishmaniasis. Moreover, in natural infection, not only do Leishmania resist humoral lytic factors and parasitize exclusively the antigen-presenting cells (APC), i. e. macrophages/dendritic cells, but also live in their phagolysosomes - a desirable site for vaccine delivery. It has been our long-term objective to safely harness the attributes of Leishmania for homing vaccines to APC to enhance their immune efficacy. Toward that end, we have genetically and chemically modified Leishmania to facilitate their loading with photosensitizers (PS), i. e. uroporphyrin (URO) and phthalocyanines (Pc), thereby rendering them photosensitive to produce ROS for cytolysis to release vaccines in the ROS-resistant phagolysosomes of APC. As shown by our recent study, Leishmania can be PS-loaded so effectively that they are susceptible to photolysis on exposure to very dim light, e. g. luciferase/luciferin-mediated luminescence (semi-auto-light). Leishmania stage-specific URO accumulation and luminescence emission are currently under study by transgenic approach for automation of Leishmania photolysis intraphagolysosomally in APCs (auto-light). Significantly, vaccination of animals with uroporphyric Leishmania followed by illumination in situ (post-light) provided initial evidence for its immuno-prophylactic activity against experimental leishmaniasis. Subsequently, photo-inactivation of Pc-loaded Leishmania (Pre-light) was shown to successfully deliver a surrogate vaccine, i. e. ovalbumin (OVA) to APC, which presented this antigen effectively to activate OVA-specific T cells in vitro and in vivo. Most recently, we obtained preliminary evidence, showing that Leishmania were photolysed to completion far more consistently when sensitized with both URO and Pc than with each individually. We now propose 2 specific aims to study this double photosensitization (DP) of Leishmania in detail under pre-, post- and auto-light conditions as follows: [1] To evaluate the safety of DP-photo-inactivated Leishmania by assessing their survival in vitro in APCs and in vivo in mice; [2] To evaluate the efficacy of DP-photo-inactivated Leishmania to deliver OVA as a surrogate vaccine to APCs for presentation to elicit T cell immune responses in vitro and in vivo. Complete photolysis of Leishmania after DP is expected not only to ascertain their elimination for safe use as a vaccine carrier but also to deliver and release vaccines effectively to increase their immune efficacy. Completion of the proposed studies is thus expected to produce a final version of these carriers optimal for photodynamic immuno-therapy & -prophylaxis not only against leishmaniasis but also other infectious/non-infectious diseases. PUBLIC HEALTH RELEVANCE: The work proposed in this application is to evaluate the safety and efficacy of a combined genetic and chemical approach to photo-inactivation of parasites. The outcome of the proposed studies will help us develop prophylactic and therapeutic photomedicines against infectious and non-infectious diseases.